The present invention is directed to processes for preparing enzymes as well as the immobilized enzyme compositions themselves, and methods for processing liquid substrates, particularly food products, by means of such immobilized enzyme compositions.
Enzymes produced by cells of plants, animals and microorganisms have long been used in food and other industrial processing for their catalytic effect.
Substantial effort has been directed to the preparation of immobilized enzyme systems, in which enzymes are converted from a water-soluble mobile state to an immobile state to improve process control and facilitate separation of the enzyme from the reactant while retaining, at least in part, their capacity for interaction with specific substrates [Kilbanov, A.M., Science, 219, 722, (1983); O. R. Zaborsky, Immobilized Enzymes, CRC Press, Cleveland, Ohio (1973), G. P. Royer, Catal. Rev. Sci. Eng. 22 (1) 29-73 (1980); J. F. Roland, "Requirements Unique to the Food and Beverage Industry", Immobilized Enzymes for Food Processing, Ed. W. H. Pitcher, CRC Press, Boca Raton, Fla. (1980)].
Food processors have had a long and continuing interest in developing the potential of enzyme systems to modify foods and beverages. In recent years, this interest has been intensified by the development of immobilization procedures that allow enzymes to be attached to solid surfaces while still retaining their functionality. Enzyme immobilization permits a high degree of process control, and reuse of the enzyme over an expanded time period. Because the enzyme no longer appears as an additive in the final product, an additional processing step to remove it in order to prevent overtreatment during storage due to continuing activity is not necessary. Major industrial-scale immobilized enzyme processes in which various amino acylases adsorbed to DEAE cellulose or DEAE agarose are employed to convert tonnage quantities of synthetic DL amino acids to the biologically available L-form. Similarly, glucose isomerase adsorbed to DEAE cellulose has been used in the full-scale conversion of corn syrup sugar to fructose.
However, there are problems arising in the utilization of immmobilized enzymes in food and beverage processing. It is, of course, not only essential that the immobilization treatment preserve enzyme activity, but it is also essential that the components of the immobilized enzyme composition and the degraded enzyme be safe for use in food products. The health and safety aspects of employing enzymes in food processing have been discussed in Enzyme Microb. Technol. 3, 105 (1981).
Enzymes have conventionally been immobilized by various methods including covalent attachment, adsorption, entrapment, crosslinking and encapsulation.
Tannic acid has been used, together with glyceraldehyde for enzyme immoblization, as described in U.S. Pat. No. 3,736,231, but its use may be restricted from food applications due to the known toxic effects of tannin in humans.
For example, U.S. Pat. No. 4,113,567 describes a modified phenolic polymer substrate for enzyme immobilization, in which the phenolic polymer has pendant aldehyde or diazonium salt groups. U.S. Pat. No. 3,767,531 concerns immobilization of an active enzyme by glutaraldehyde on a substrate of phenol formaldehyde resin. U.S. Pat. Nos. 3,992,329 and 4,078,970 relate to adsorption of an enzyme such as glucose isomerase within the pores of an inorganic support, or a porous anion exchange resin. U.S. Pat. Nos. 3,852,496 and 4,016,293 concern a process for using insolubilized lactase to hydrolyze lactose from cheese whey, in which the enzyme is treated with glutaraldehyde, silane coupling agents or other materials. U.S. Pat. No. 4,338,398 describes the cross-linking of starch degrading enzymes with a wide variety of mono and polyfunctional materials such as aldehydes, isocyanates and methylol groups, and their absorption onto a water insoluble, porous substrate.
As indicated, in order to carry out these immoblization procedures, a wide variety of chemical agents, insoluble supports and solvents have been employed, but most have substantial disadvantages for use in systems intended for food processing utilization. For example, these systems which rely on cyano- or halogenated chemical derivatives, aldehydes, isocyanates, carbodiamides, diazo compounds, phosgene derivatives and the like for enzyme attachment, spacing or cross liking are undesirable for use in food processing because of the toxic potentials of these compounds. The use of glutaraldehyde as an effective cross-linking agent for enzymes has been recognized for many years. However, commercial solutions may contain undesirable quantities of substances such as acrolein, glutaric acid and glutaraldoxine and the continuing development of biological data in respect to glutaraldehyde may indicate undesirable properties for food processing use. It is apparent that the presence of these agents in enzyme modified foodstuffs, due to degradation, would constitute a serious health hazard. Therefore, it is important to find systems for immobilizing enzymes in which all of the components are recognized as safe and are also inexpensive.
Accordingly, it is an object of the present invention to provide processes for preparing insolublized enzyme compositions which utilize food grade materials which are generally recognized as safe for human consumption. It is a further object to provide active immobilized enzyme compositions prepared from edible components and having extended useful life for enzymatic processing. An additional object is the provision of enzymatic food processing processes utilizing such compositions.